Pharmacia U.S.E. Mutagenesis

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Jan 27 13:44:05 EST 1994


wetsel_r at wums.wustl.edu writes:

: sschendel at bilbo.bio.purdue.edu (Sharon Schendel) wrote:

: Just a short note concerning U.S.E. Mutagenesis, in particular, Pharmacia's
: kit.  I had been trying to make mutants using Stratagene's pSKBluescript for my
: vector.  The kit's manual makes the claim that plasmids which have lost the
: unique restriction site have a 90% chance of gaining the desired mutation.  I
: found three such plasmids, but upon sequencing, found that they had only lost
: the selection site... they had not gained my mutation.

I don't know very much about this kit, but I assume it is a double primer
site-specific mutagenesis system. I did a similar thing with pSelect1. After
sequencing 12 clones, I found they were all identical. I think you may have
gotten three sibling transformants.  Maybe isolating more than three would give
you a much better chance of finding the mutant. Just a horrible thought
though...make sure the oligos are on the same strand :-)

: Pharmacia's technical service rep informed me that for some "unknown reason"
: pSKBluescript will not work with their kit.  Those plasmids which they could
: guarantee would work are pBR322 and the pUC vectors (i.e. those marketed by
: Pharmacia).  Just thought that I'd share this, since there is no mention of
: plasmids that would not work with the U.S.E. method either in Pharmacia's
: manual or in the original Deng and Nickoloff paper (Anal.  Biochem 200,81).

Sure...no guarantees, no money back! ;-)

: Sharon Schendel
: sschendel at bilbo.bio.purdue.edu

> This is bizzare Sharon...  From what I could tell, I was under the 
> assumption that pBluescript was DERIVED from pUC19.  So in theory, it 
> should work.  I know someone out there will ask for my source but I 
> distinctly remember reading that the backbone of pBS was initialy derived 
> from pUC19 and then S_Gene added additional restriction sites, and the f1, 
> but the ori, B-gal, and amp all came from puc.
>
>
> Anyone correct if I'm mistaken but I'll also check again myself.
> David
> haviland at kids.wustl.edu

Yes, you are right. There is no reason why this should not work
because pBluescript is derived from pUC19:

@article{Short1988,
author = "J. M. Short
     and J. M. Fernandez
     and J. A. Sorge
     and W. D. Huse",
title = "$\lambda$ {ZAP}: a bacteriophage $\lambda$ expression
vector with {\em in vivo} excision properties",
journal = "Nucleic Acids Res.",
volume = "16",
number = "15",
pages = "7583-7600",
note = "construction of pBluescript",
year = "1988"}

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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